(1) Field of the Invention
The present invention relates to an NMR (nuclear magnetic resonance) apparatus, in particular, to an apparatus of nuclear magnetic resonance measurement by using circulation flow for sample condition control which apparatus is for carrying out measurement repeatedly while changing the measuring conditions of a sample in the NMR apparatus.
(2) Description of the Related Arts
Molecules such as proteins having certain functions within a living body have a larger molecular weight than that of compounds used for medicines or the like and have properties as a large molecule.
Large molecules typified by proteins have a molecular function in a solution thereof and this function is inhibited or accelerated when they bind to a certain small molecule. There are many methods for detecting binding of a large molecule to a certain small molecule or interaction therebetween. In particular, in measurement (which will hereinafter be called “NMR measurement”) utilizing nuclear magnetic resonance permitting direct observation of the information of a molecular structure of the large molecule or small molecule, it is possible to analyze, from a change in the measurement spectrum of the concentration of the molecule, not only a dissociation constant between the large molecule and low molecule or reaction rate of them but also interaction of them based on the structure of the molecules. Refer to, for example, the description in International Publication No. WO01/023889 (Japanese Patent Application No. 2001-527224) with regard to a method of measuring the interaction between a protein and a low molecule by means of NMR.
Of large molecules to be used for NMR measurement, proteins are produced, for example, by extraction from naturally occurring living organisms, extraction from an abundant expression system making use of Escherichia coli or the like having, incorporated therein, a gene related to the formation of a protein, or by a method utilizing a non-cell expression system causing abundant expression of a protein without using a living cell. Labeling of hydrogen, carbon and nitrogen, which are main elements constituting a protein, with a radioisotope is sometimes carried out. This labeling includes labeling of a combination of three elements, that is, hydrogen, carbon and nitrogen constituting a protein, comprehensive labeling method for labeling all them, and selective labeling method of labeling only atoms belonging to a specific amino acid residue. In any method, a labeling cost is high.
A nuclear magnetic resonance spectrometer (which will hereinafter be called NMR apparatus) has typically a magnet for causing a magnetostatic field B0 and a nuclear magnetic resonance probe located at a bore portion inside of this magnet. This nuclear magnetic resonance probe includes at least one coil for applying an RF magnetic field B1 to a sample of interest and detecting reaction (response) of the sample to the magnetic field thus applied.
Conventional nuclear magnetic resonance probes include probes for the static setting sample and flow through probes. In probes for the static setting sample, the sample is put in a glass tube or ampoule (which will hereinafter be called “sample tube”) and measurement is started after setting this sample tube at a predetermined position of the NMR apparatus.
In conventional probes for the static setting sample, a change in the NMR spectrum due to an increase in the concentration can be observed by carrying out NMR measurement while titrating a small molecule in a sample tube having an opening portion.
Conventional flow through probes each has a sample inlet, a sample outlet, and an internal tubular portion extending between the inlet and outlet. The internal tubular portion includes a cell for retaining a sample therein. The sample is put in this probe from the sample inlet, flows in the internal tubular portion, and enters the cell. After measurement, it flows in the tubular portion and is discharged out of the probe.
A combination of the conventional flow through probe and a robot-using sample transfer system has been used. Various sample transfer systems are employed for this combination and one of them is commercially available from Gilson, Inc. In such a system, samples adjusted for a plurality of measurement conditions must be prepared and put in a plurality of containers in advance, respectively. The samples are each fed to a predetermined flow through probe after passing through an apparatus capable of taking it out from the container. Each sample is discharged out of the probe after completion of the NMR measurement. Refer to International Publication No. WO2003/007009 (Japanese Unexamined Patent Application Publication No. 2004-534958) with regard to a preferred conventional system for transferring the sample to the probe.